大鼠腰骶髓和延髓星形胶质细胞及神经元对慢性结肠炎的反应

目的探讨大鼠腰骶髓和延髓星形胶质细胞及神经元对慢性结肠炎的反应,及反应性星形胶质细胞和反应性神经元之间的关系.方法成年雄性SD大鼠,实验组（n=17）给予三硝基苯磺酸（TNBS）灌肠诱导结肠炎;对照组（n=16）给予生理盐水灌肠.免疫组织化学法显示大鼠腰骶髓和延髓内胶质原纤维酸性蛋白（GFAP）阳性星形胶质细胞和Fos阳性神经元.结果TNBS灌肠后,GFAP阳性星形胶质细胞主要分布在脊髓背角浅层（Ⅰ～Ⅱ层）、中间外侧核（Ⅴ层）、后连合核（Ⅹ层）和腹角外侧核（Ⅸ层）.Fos阳性神经元集中分布在背角深层（Ⅲ～Ⅳ,Ⅴ～Ⅵ层）.在延髓,两者均主要分布在由孤束核、中间网状带和腹外侧区组成的延髓内脏带（MVZ）.TNBS灌肠后3、7、14 d,脊髓中GFAP阳性细胞密度明显高于对照组（P＜0.05）.TNBS灌肠后3 d,延髓中GFAP阳性细胞密度明显高于对照组（P＜0.05）.TNBS灌肠后28 d,脊髓和延髓中GFAP阳性细胞密度下降,与对照组无显著性差异（P＞0.05）.结论结肠炎性刺激引起脊髓和延髓中星形胶质细胞激活.随着结肠炎的恢复,星形胶质细胞的反应性下降.在延髓内脏带,反应性星形胶质细胞与反应性神经元关系密切.     

大鼠前脑星形胶质细胞和神经元对一侧胫、腓骨骨折的反应及其相互关系

目的　研究大鼠前脑内星形胶质细胞 (ASs)及神经元 (Ns)对一侧胫、腓骨骨折的反应及相互关系。方法　应用细胞免疫组织化学方法观察前脑内胶质原纤维酸性蛋白 (GFAP)、Fos蛋白以及酪氨酸羟化酶 (TH)在左侧胫、腓骨骨折后的表达变化。　结果　 1 一侧胫、腓骨骨折后 ,前脑GFAP阳性表达的ASs有明显的核团定位。在缰外侧核内侧区 (LHb)、下丘脑室旁核 (Pa)、视上核 (SON)、视交叉上核 (SCh)、终纹床核 (BST)、杏仁中央核 (Ce)和内侧杏仁核 (Me)及皮层等脑区内均可观察到有GFAP阳性ASs分布 ,且两侧分布无明显差异。 2 Fos阳性Ns的分布与GFAP阳性ASs上述分布部位基本一致 ,两者关系密切。 3 Pa等部位有大量Fos TH双标Ns,四周是密集的GFAP阳性ASs包围 ,形成以神经元为中心 ,周围包绕ASs,共同构成神经元 星形胶质细胞复合体 (N ASC)。　结论　上述核团的ASs参与了下肢骨折伤害性刺激的应激反应及其调节过程 ,且与Ns关系密切 ,可能主动地影响Ns的活动。     

大鼠腰髓背角星形胶质细胞对外伤性疼痛刺激的反应及其与神经元关系的免疫电镜研究

本研究采用抗缝隙连接蛋白 43 ( Cx43 )和抗缝隙连接蛋白 3 2 ( Cx3 2 )免疫电镜双标记方法 ,观察了一侧胫、腓骨骨折后大鼠腰髓背角星形胶质细胞与神经元的超微结构改变。结果发现 ,在脊髓背角星形胶质细胞与神经元之间的结合区域存在着如下的超微结构 :( 1)轴突终末直接与星形胶质细胞突起 ( Cx43阳性 )形成一种突触样结构 ;( 2 )由神经元突触前膜、突触后膜及星形胶质细胞突起形成的“三方突触结构”;( 3 )星形胶质细胞与星形胶质细胞之间的缝隙连接 ;( 4 )新发现的由一侧的神经元( Cx3 2阳性 )和另一侧的星形胶质细胞突起 ( Cx43阳性 )组成的一种超微结构 ,骨折后此结构数目显著增高 ,可能是缝隙连接的同源结构 ,暂称为异源性缝隙连接。结果提示 :异源性缝隙连接结构可被疼痛刺激激活 ,并极有可能是神经元与星形胶质细胞之间密切接触并进行物质交流的结构基础之一     

低渗刺激后大鼠视上核内星形胶质细胞和神经元的可塑性改变及其相互关系

为观察低渗刺激后大鼠视上核(SON)内星形胶质细胞和神经元的可塑性反应及其两者在反应时间和空间上的相互关系,我们采用大鼠尾静脉注射 5. 5ml/kg低渗盐水(0. 83% 葡萄糖+ 0. 3% NaCl)制作模型,应用抗Fos、抗胶质原纤维酸性蛋白(GFAP)单标记,以及抗Fos/抗GFAP双标记、抗Fos/抗血管加压素 (VP) /抗GFAP三标记的免疫组化方法。结果发现:在SON中GFAP阳性星形胶质细胞数量在刺激后 15min增加,其胞体肥大,突起伸长变粗, 45min达高峰。Fos阳性星形胶质细胞胞核呈蓝黑色小点,刺激后 15~45min达高峰, 90min明显减少;Fos阳性神经元胞核较大,刺激后 15min未见表达, 45min少量表达, 90min表达增加。该结果表明低渗刺激后,SON内星形胶质细胞的Fos表达早于神经元,三重标记显示星形胶质细胞与神经元关系密切,提示SON内星形胶质细胞可能在低渗刺激的调节中发挥一定作用。     

脂多糖对大鼠中脑和桥脑内Fos、GFAP、OX42表达的影响

目的 探讨单次腹腔注射脂多糖(LPS),中脑和桥脑神经元、星形胶质细胞和小胶质细胞的可塑性变化。方法 抗Fos蛋白、抗胶质原纤维酸性蛋白(GFAP)、抗特异性标记小胶质细胞(OX42)和抗Fos／GFAP双重免疫组织化学标记法。结果 Fos阳性神经元分布于上丘、中脑导水管周围灰质、臂旁核和蓝斑。Fos蛋白在注射后30min。表达,1～3h为高峰。GFAP阳性星形胶质细胞胞体变大,突起增粗,细胞密度增加,30min出现表达,1h为高峰,3h后减少。OX42阳性小胶质细胞首先于脑室周围灰质表达,注射后6h达到高峰,胞体变大,全脑分布。相应于Fos阳性神经元分布区域,GFAP阳性星形胶质细胞和OX42阳性小胶质细胞深染和密集。结论 上丘、臂旁核、蓝斑内神经元、星形胶质细胞、小胶质细胞可能参与神经免疫调节,臂旁核可能为此调节通路的中继站之一。     

大鼠侧脑室注射甘珀酸后下丘脑视上核星形细胞和神经元对高渗刺激反应的影响

目的 观察大鼠侧脑室注射甘珀酸后下丘脑视上核星形细胞和神经元对高渗刺激反应的影响。方法 经侧脑室注射缝隙连接阻断剂甘珀酸(carbenoxolone,CBX)及尾静脉注射9％NaCl溶液后，用放射性免疫分析法测定大鼠血浆中加压素(VP)含量，免疫组织化学方法观察下丘脑视上核(SON)神经元的：Fos和星形细胞的Fos以及胶质原纤维酸性蛋白(GFAP)表达的变化。结果 1．未注射CBX组大鼠在高渗刺激后45min，血浆中VP的含量明显升高；SON中观察到GFAP／Fos阳性的星形细胞和Fos阳性神经元；2．GFAP／Fos阳性星形细胞高峰的出现早于Fos阳性神经元；3．向侧脑室注射CBX2h后，经尾静脉注射9％NaCl溶液，45Bin后血浆中的VP含量未升高，SON中GFAP／Fos阳性星形细胞的表达与未注射CBX组相同，Fos阳性神经元则显著减少。结论 SON的星形细胞对高渗刺激发生反应早于神经元，并可能通过缝隙连接影响神经元对渗透压的调节功能。     

大鼠三叉神经尾侧亚核内星形胶质细胞对疼痛刺激的反应及与神经元的关系

目的 研究大鼠三叉神经尾侧亚核(Sp5C)星形胶质细胞对唇下注射福尔马林所致疼痛的反应及其与神经元的关系。方法 用免疫组织化学方法，显示注射后不同时间Sp5C星形胶质细胞与神经元内抗磷脂酶C(PLC)、抗Fos和抗胶质原纤维酸性蛋白(GFAP)免疫组织化学产物的表达和分布。结果 正常大鼠Sp5C无免疫组织化学阳性染色，唇下注射福尔马林后，Sp5C内的星形胶质细胞出现抗PLC、抗Fos和抗GFAP阳性染色，神经元出现抗PLC和抗Fos阳性染色，且有相同的亚核分布，关系密切。抗PLC和抗Fos免疫组织化学阳性先出现于星形胶质细胞，而后在神经元出现表达。结论 Sp5C内星形胶质细胞可能参与中枢神经系统对疼痛刺激的调节，并主动调节神经元的活动。     

皮质酮对大鼠脊髓背角星形胶质细胞活动的影响

目的:探讨皮质酮(corticosterone,CORT)对培养的大鼠脊髓背角星形胶质细胞活性的调节作用。方法:培养纯化新生SD大鼠脊髓背角星形胶质细胞,荧光双重标记技术检测培养的星形胶质细胞糖皮质激素受体(glucocorticoid receptor,GR)及胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)共表达;免疫印迹技术检测星形胶质细胞GFAP表达的变化;高效液相色谱法(high performance liquid chromatography,HPLC)检测星形胶质细培养液中谷氨酸含量。结果:培养的脊髓背角星形胶质细胞均表达GR;CORT孵育3 h可降低脊髓背角星形胶质细胞GFAP表达,GR拮抗剂RU38486可阻断CORT的作用;但CORT对星形胶质细胞谷氨酸释放无显著影响。结论:CORT可降低脊髓背角星形胶质细胞GFAP表达,但对谷氨酸释放无显著影响。     

ATP通过P2Y1受体引发脊髓背角星形胶质细胞[Ca2+]i升高和GFAP表达上调

目的 观察体外培养的背角星形胶质细胞P2Y1受体激活对其[Ca2 ]i的变化和GFAP表达的影响.方法 培养并纯化脊髓背角星形胶质细胞,采用免疫组织化学染色观察背角星形胶质细胞P2Y1受体及GFAP的表达,激光共聚焦技术观察星形胶质细胞[Ca2 ]i的变化.结果 体外培养的大鼠脊髓背角星形胶质细胞大多表达P2Y1受体;P2Y受体激动剂ATP、ADP、ADP-βs剂量依赖性促进星形胶质细胞[Ca2 ]i升高;10 μg/mL的ATP、ADP和ADP-βs显著增加胞内[Ca2 ]i,此作用可被特异性P2Y1受体拮抗剂MRS2179所阻断,并具量效关系.免疫组织化学染色结果显示,100 μg/mL的ATP、ADP和ADP-βs作用下,星形胶质细胞GFAP表达上升,此效应可被100 μg/mL的MRS2179所抑制.结论 体外培养的大鼠脊髓背角星形胶质细胞表达P2Y1受体;P2Y1受体介导了ATP、ADP及ADP-βs促进星形胶质细胞[Ca2]i升高和GFAP表达增强的过程.     

缺血再灌注对大鼠神经元与星形胶质细胞的影响

应用免疫组织化学单标记法分别观察大鼠在大脑中动脉阻塞再灌注时胶质原纤维酸性蛋白(GFAP)和Fos蛋白在大脑皮质内表达的时间规律,并用免疫组织化学双重标记法观察GFAP和Fos蛋白表达的相互关系。结果发现在缺血1h再灌注2h时,大脑皮层的星形胶质细胞被激活,细胞体积增大,突起粗大,呈GFAP阳性。星形胶质细胞的反应直至48h依然强烈。被激活的星形胶质细胞和神经元表达Fos蛋白,并呈现时程变化规律。结果提示星形胶质细胞可能和神经元一起参与了大脑皮层缺血再灌注后的变化。     

热应激对大鼠脑桥臂旁核Fos和GFAP表达的影响

为了探讨急性热应激(heat stress,HS)后大鼠脑桥臂旁核Fos蛋白及胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达变化,本实验将大鼠置于恒温恒湿温度舱内,舱内温度调至24℃、34℃、38.5℃或42℃、湿度为60%,建立HS大鼠模型,1h后结束刺激,立即断头取脑,应用免疫组织化学染色方法检测脑桥臂旁核内Fos蛋白和GFAP的表达情况。结果显示:脑桥臂旁核内可见大量的Fos样免疫阳性神经元和GFAP样免疫阳性的星形胶质细胞,其中在24℃~38.5℃组Fos蛋白的表达随温度增加而增加,38.5℃组为高峰,而在42℃组Fos蛋白表达又有所减少。GFAP样免疫阳性星形胶质细胞在34℃组出现胞体变大,突起增粗的现象,且细胞数量增加,38.5℃组最显著,但在42℃组星形胶质细胞的数量则有所减少,并出现胞体形态不规则、突起断裂的现象。本实验结果表明脑桥臂旁核参与热应激反应的神经免疫调节,可能为此调节通路的中继站之一。     

Increased formalin-induced pain and expression of fos neurons in the lumbar spinal cord of prenatally stressed infant rats

When pregnant dams are stressed, there is a resultant alteration in brain development and behavior in their offspring. Prior work has shown increased nociceptive responses in adolescent or adult rats born of stressed dams. However, the age at which those changes first occur is not known. The aim of the present study was to evaluate the effect of prenatal stress on pain sensitivity in the formalin test in 7-day-old rats, behaviorally and by fos-like immunoreactivity (Fos-LI) in the lumbar spinal cord dorsal horn. The behavioral response to intraplantar injection of formalin is represented by two nociceptive phases separated by an interphase during which nociceptive responses decrease; the interphase is not seen until the start of the third postnatal week and appears as descending inhibitory monoaminergic systems develop. Prenatally stressed infants showed increased nociceptive responses in the second, tonic phase and a large increase in the number of formalin-induced Fos-LI neurons in the lumbar dorsal horn, a result consistent with the behavioral data. The increased nociception in prenatally stressed 7-day-old pups may be associated with the decrease in the intensity of serotonin-like immunoreactivity and density of serotonergic cells in the lumbar spinal cord dorsal horn and the dorsal raphe nucleus reported earlier.     

大鼠脊髓背角内FOS和NDP阳性神经元的分布与联系(英文)

本文应用还原性尼克酰胺腺嘌呤二核苷酸脱氢酶（NDP）组织化学和原癌即刻早期基因c-fos表达产物Fos免疫细 胞化学方法，观察了大鼠脊髓背角内 NDP和 Fos阳性神经元的分布与联系。一侧足跖部皮下注射福尔马林后，同侧背角内可见大量Fos阳性细胞，而对侧背角内未见或偶见Fos阳性细胞。在背角各层中，大多数Fos阳性细胞分布于Ⅰ层及Ⅱ层外带的内侧部．背角内也可见大量NDP阳性胞体，纤维和终末，密集分布于Ⅱ层内带。双标结果说明部分背用内的Fos阳性细胞也是NDP阳性，双标神经元主要分布于区层的内侧部。在Ⅱ层内，Fos阳性细胞周围常有NDP阳性纤维和终末分布，部分NDP阳性终未直接附着干Fos阳性细胞膜上。本文结果为NO参与脊髓内伤害性刺激信息传递过程提供了形态学证据。     

大鼠脊髓内囊泡膜谷氨酸转运体与星形胶质细胞之间的联系(英文)

本研究应用双重免疫荧光组织化学法观察了大鼠脊髓胶质细胞内囊泡膜谷氨酸转运体 (VGlu Ts包括 VGlu T1,VG-lu T2及 VGlu T3 )的表达状况。结果显示 :大鼠脊髓内 VGlu T1、VGlu T2和 VGlu T3样阳性轴突终末与星形胶质细胞之间形成密切接触 ;部分星形胶质细胞同时呈 VGlu T3样免疫阳性。上述结果提示 ,脊髓内谷氨酸能神经终末与星形胶质细胞之间存在着信号传递 ;且在星形胶质细胞通过胞吐作用释放谷氨酸的过程中 VGlu T3可能发挥重要作用。     

The lumbar spinal cord glial cells actively modulate subcutaneous formalin induced hyperalgesia in the rat

We investigated the response and relationship of glial cells and neurons in lumbar spinal cord to hyperalgesia induced by the unilateral subcutaneous formalin injection into the hindpaw of rats. It was demonstrated that Fos/NeuN immunoreactive (-IR) neurons, glial fibrillary acidic protein (GFAP)-IR astrocytes and OX42-IR microglia were distributed in dorsal horn of lumbar spinal cord, predominantly in the superficial layer. In the time-course studies, GFAP-IR astrocytes were firstly detected, OX42-IR microglia were sequentially observed, Fos/NeuN-IR neurons were found slightly late. Immunoelectron microscopy studies established that many heterotypic gap junctions (HGJs), which consisting of Cx43-IR astrocytic process on one side and Cx32-IR dendrite on the other side, were present in superficial layer of dorsal horn. Ninety-one HGJs were found in 100 areas of experimental rats and occupied 91%, while only 39% HGJs were found in control rats. In experimental rats pretreated with intrathecal (i.t.) application of the carbenoxolone (a gap junction blocker) or fluorocitrate (a glial metabolic inhibitor), the paw withdrawal thermal latency was prolonged than those application of the sterile saline (i.t.). It suggests that spinal cord glial cells may play an important role for modulation of hyperalgesia induced by noxious stimuli through HGJs which located between astrocytes and neurons.     

糖皮质激素对甲醛致炎大鼠c-fos表达的影响

目的为糖皮质激素(GCS)对神经细胞的作用提供形态学资料。方法30只SD大鼠随机分为:对照组、单纯甲醛组、地塞米松预处理组、RU486预处理组、RU486+地塞米松预处理组,甲醛刺激后采用动物行为学方法、免疫组织化学法、神经细胞染色和形态计量学方法进行观察。结果在大鼠后爪掌面皮下注射稀释甲醛20μl可造成注射处急性持续性炎症。1h后,同侧脊髓腰膨大背角浅层。双侧中缝大核、丘脑、大脑皮层Fos免疫阳性神经元(FLI)总数明显上升,对侧脊髓内少量散在分布。用地塞米松预处理2h后可使相应部位FLI神经元数明显降低,具有剂量依赖性。外周水肿。疼痛表现与同侧背角浅层FLI细胞数呈正相关。用糖皮质激素受体阻断剂RU486可部分反转GCS的效应。结论伤害性刺激可引起中枢神经系统c-fos广泛表达。GCS可降低脊髓后角神经元的兴奋性。     

Induction of microglial and astrocytic response in the adult rat lumbar spinal cord following middle cerebral artery occlusion

The response of microglia and astrocytes, as detected immunohistochemically by the monoclonal antibody OX-42 and anti-glial fibrillary acidic protein (GFAP) respectively, was studied in the rat lumbar spinal cord following focal cerebral ischaemia produced by permanent occlusion of the middle cerebral artery (MCA) above the rhinal fissure. At 1 and 2 days after right-sided MCA occlusion, OX-42 immunoreactivity of microglia in both the contralateral dorsal and ventral horns of the lumbar spinal cord was moderately increased compared with cells of the ipsilateral side. The microglial reaction was progressive, with some cells transformed into amoeboid form considered to be macrophages at day 3. By 5 days, many of the reactive microglia, notably in the ventral horn, appeared to encircle the soma of motoneurons. At 7 days, the microglial reaction had subsided while astrocytes in the same area were hypertrophied to replace the perineuronal microglia. The microglial response in both the cerebral cortex and lumbar spinal cord was effectively reduced by the N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801. Present results suggest that following MCA occlusion, the vigorous response of microglia, and subsequently astrocytes, in the spinal cord in extra-focal areas far removed from the primary site of ischaemia may be mediated by glutamate released from the ischaemic corticospinal neurons through NMDA receptors on the postsynaptic spinal cord neurons. Received: 21 March 1997 / Accepted: 23 June 1997     

Inhibition of spinal cytosolic phospholipase A(2) expression by an antisense oligonucleotide attenuates tissue injury-induced hyperalgesia

Activation of the spinal phospholipase A(2) (PLA(2)) -cyclooxygenase (COX) -prostaglandin signaling pathway is widely implicated in nociceptive processing. Although the role of spinal COX isoforms in pain signal transmission has been extensively characterized, our knowledge of PLA(2) enzymes in this cascade is limited. Among all PLA(2) groups, cytosolic calcium-dependent PLA(2) group IVA (cPLA(2)IVA) appears to be the predominant PLA(2) enzyme in the spinal cord. In the present study we sought to (i) characterize anatomical and cellular distribution and localization of cPLA(2)IVA in dorsal horn of rat spinal cord, (ii) verify efficacy and selectivity of intrathecal (IT) delivery of an antisense oligonucleotide (AS) targeting rat cPLA(2)IVA mRNA on spinal expression of this enzyme, and (iii) examine the effect of down-regulation of spinal cPLA(2)IVA on peripheral tissue injury-induced pain behavior. Here we demonstrate that cPLA(2)IVA is constitutively expressed in rat spinal cord, predominantly in dorsal horn neurons and oligodendrocytes but not in astrocytes or microglia. Intrathecal injection of AS significantly down-regulated both protein and gene expression of cPLA(2)IVA in rat spinal cord, while control missense oligonucleotide (MS) had no effect. Immunocytochemistry confirmed that the reduction occurred in neurons and oligodendrocytes. cPLA(2)IVA AS did not alter expression of several other PLA(2) isoforms, such as secretory PLA(2) (groups IIA and V) and calcium-independent PLA(2) (group VI), indicating that the AS was specific for cPLA(2)IVA. This selective knockdown of spinal cPLA(2)IVA did not change acute nociception (i.e. paw withdrawal thresholds to acute thermal stimuli and intradermal formalin-induced first phase flinching), however, it significantly attenuated formalin-induced hyperalgesia (i.e. second phase flinching behavior), which reflects spinal sensitization. Thus the present findings suggest that cPLA(2)IVA may specifically participate in spinal nociceptive processing.